Ischemic stroke seriously threatens human health because of high rates of morbidity, mortality and disability. This study compared the effects of nicotinamide adenine dinucleotide (NAD) and butylphthalide (NBP) on and ischemic stroke models. Transient middle cerebral artery occlusion/reperfusion (t-MCAO/R) model was established in mice, and the cultured primary cortical neurons were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R). Cerebral infarct volume, neurobehavioral indices, antioxidant activity, ATP level and lactic acid content were determined. The neuroprotective effects of NAD or NBP were compared using sirtuin inhibitor niacinamide (NAM). Intraperitoneal injection of NBP within 4 h or intravenous injection of NAD within 1 h after t-MCAO/R significantly reduced the volume of infarcts, cerebral edema, and neurological deficits. Administration of NAD and NBP immediately after t-MCAO/R in mice showed similar neuroprotection against acute and long-term ischemic injury. Both NAD and NBP significantly inhibited the accumulation of MDA and HO and reduced oxidative stress. NAD was superior to NBP in inhibiting lipid oxidation and DNA damage. Furthermore, although both NAD and NBP improved the morphology of mitochondrial damage induced by ischemia/reperfusion, NAD more effectively reversed the decrease of ATP and increase of lactic acid after ischemia/reperfusion compared with NBP. NAD but not NBP treatment significantly upregulated SIRT3 in the brain, but the sirtuin inhibitor NAM could abolish the protective effect of NAD and NBP by inhibiting SIRT1 or SIRT3. These results confirmed the protective effects of NAD and NBP on cerebral ischemic injury. NBP and NAD showed similar antioxidant effects, while NAD had better ability in restoring energy metabolism, possibly through upregulating the activity of SIRT1 and SIRT3. The protection provided by NBP against cerebral ischemia/reperfusion may be achieved through SIRT1.